rs-504393 and Disease-Models--Animal

There have been several studies on the role of the monocyte chemotactic protein-1/C-C chemokine receptor type 2 (CCR2) signalling pathway in fibrotic diseases, which identified the blockade of this pathway as a potential therapeutic target for treating fibrosis. We examined the efficacy of CCR2 antagonist (RS-504393) in a mouse model of scleroderma induced by bleomycin. RS-504393 was administered via intradermal injection 6 hours prior to bleomycin injection, in the same sites. Histopathological examination showed that RS-504393 treatment suppressed dermal fibrosis and decreased dermal thickness. The numbers of mast cells and myofibroblasts in the skin of RS-504393-treated mice were significantly lower compared with those in PBS-treated mice. Moreover, the amount of collagen in the skin of RS-504393-treated mice was significantly lower compared with that in the PBS-treated mice. Additionally, mRNA levels of TGF-β1 and collagen I alpha 1 in sclerotic skin were significantly decreased by RS-504393, and semiquantitative histopathological scoring of the lungs showed inhibition of fibrosis in RS-504393-treated mice. The amount of collagen in the lung of the RS-504393-treated mice was lower compared with that in the PBS-treated mice. These data suggest that CCR2 antagonist RS-504393 may be a therapeutic agent for human scleroderma.

Topics: Animals; Benzoxazines; Bleomycin; Cell Count; Collagen; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Female; Fibrosis; Lung; Mast Cells; Mice; Myofibroblasts; Receptors, CCR2; RNA, Messenger; Scleroderma, Systemic; Skin; Spiro Compounds; Transforming Growth Factor beta1

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Lymphocyte antigen 6Chigh (Ly-6C

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, Ly; Benzoxazines; Bone Marrow; Cell Movement; Chemokine CCL2; Disease Models, Animal; Humans; Imidazoles; Lung; Mice; Monocytes; p38 Mitogen-Activated Protein Kinases; Pyridines; Receptors, CCR2; Spiro Compounds; Tidal Volume; Ventilator-Induced Lung Injury; Ventilators, Mechanical

Approximately one-third of individuals with sickle cell disease (SCD) develop chronic pain. This debilitating pain is inadequately treated because the underlying mechanisms driving the pain are poorly understood. In addition to persistent pain, patients with SCD are also in a tonically proinflammatory state. Previous studies have revealed that there are elevated plasma levels of many inflammatory mediators including chemokine (c-c motif) ligand 2 (CCL2) in individuals with SCD. Using a transgenic mouse model of SCD, we investigated the contributions of CCL2 signaling to SCD-related pain. Inhibition of chemokine receptor 2 (CCR2), but not CCR4, alleviated the behavioral mechanical and cold hypersensitivity in SCD. Furthermore, acute CCR2 blockade reversed both the behavioral and the in vitro responsiveness of sensory neurons to an agonist of TRPV1, a neuronal ion channel previously implicated in SCD pain. These results provide insight into the immune-mediated regulation of hypersensitivity in SCD and could inform future development of analgesics or therapeutic measures to prevent chronic pain.

Topics: Anemia, Sickle Cell; Animals; Benzoxazines; Cryopyrin-Associated Periodic Syndromes; Disease Models, Animal; Hyperalgesia; Mice; Mice, Transgenic; Receptors, CCR2; Sensory Receptor Cells; Spiro Compounds

Neuroinflammation and microglial activation have been implicated in both alcohol use disorders (AUD) and fetal alcohol spectrum disorders (FASD). Chemokine monocyte chemoattractant protein 1 (MCP-1) and its receptor C-C chemokine receptor type 2 (CCR2) are critical mediators of neuroinflammation and microglial activation. FASD is the leading cause of mental retardation, and one of the most devastating outcomes of FASD is the loss of neurons in the central nervous system (CNS). The underlying molecular mechanisms, however, remain unclear. We hypothesize that MCP-1/CCR2 signaling mediates ethanol-induced neuroinflammation and microglial activation, which exacerbates neurodegeneration in the developing brain.. MCP-1/CCR2 signaling played an important role in ethanol-induced microglial activation/neuroinflammation and neurodegeneration in the developing brain. The effects may be mediated by the interaction among MCP-1/CCR2 signaling, TLR4, and GSK3β.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Benzoxazines; Brain; Caspase 3; Cells, Cultured; Central Nervous System Depressants; Chemokine CCL2; Disease Models, Animal; Encephalitis; Ethanol; Gene Expression Regulation, Developmental; Indazoles; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Neurodegenerative Diseases; Propionates; Receptors, CCR2; Signal Transduction; Spiro Compounds

Neuroinflammation induced by peripheral trauma plays a key role in the development of postoperative cognitive dysfunction (POCD). Substantial evidence points to reactive glia as a pivotal factor during the inflammation process. However, little is known about the functional interactions between astrocytes and microglia. Recent evidence suggests the involvement of the CCL2-CCR2 pathway in CNS inflammation-related diseases. Our previous studies have suggested that astrocyte-derived CCL2 can induce microglial activation in vitro. Within this context, we sought to determine if the CCL2/CCR2 axis is involved in the crosstalk between astrocytes and microglia, contributing to increased neuroinflammation. Here, we show that tibial fracture surgery promoted CCL2 upregulation in activated astrocytes, increased CCR2 expression in activated microglia, and induced deficits in learning and memory. Site-directed pre-injection of RS504393, a CCR2 antagonist, inhibited this effect by reducing microglial activation, M1 polarization, inflammatory cytokines, and neuronal injury and death and improving cognitive function. Taken together, these data implicate CCL2-CCR2 signaling in astrocyte-mediated microglial activation in central nervous system (CNS) inflammation and suggest that interference with CCL2 signaling could constitute another potential therapeutic target for POCD.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; Benzoxazines; Chemokine CCL2; Cognitive Dysfunction; Disease Models, Animal; Hippocampus; Inflammation; Male; Microglia; Neurons; Nootropic Agents; Postoperative Complications; Random Allocation; Rats, Sprague-Dawley; Receptors, CCR2; Signal Transduction; Spiro Compounds; Tibial Fractures

We previously found in our embryonic studies that proper regulation of the chemokine CCL12 through its sole receptor CCR2, is critical for joint and growth plate development. In the present study, we examined the role of CCR2 in injury-induced-osteoarthritis (OA).. We used a murine model of injury-induced-OA (destabilization of medial meniscus, DMM), and systemically blocked CCR2 using a specific antagonist (RS504393) at different times during disease progression. We examined joint degeneration by assessing cartilage (cartilage loss, chondrocyte hypertrophy, MMP-13 expression) and bone lesions (bone sclerosis, osteophytes formation) with or without the CCR2 antagonist. We also performed pain behavioral studies by assessing the weight distribution between the normal and arthritic hind paws using the IITS incapacitance meter.. Testing early vs delayed administration of the CCR2 antagonist demonstrated differential effects on joint damage. We found that OA changes in articular cartilage and bone were ameliorated by pharmacological CCR2 blockade, if given early in OA development: specifically, pharmacological targeting of CCR2 during the first 4 weeks (wks) following injury, reduced OA cartilage and bone damage, with less effectiveness with later treatments. Importantly, our pain-related behavioral studies showed that blockade of CCR2 signaling during early, 1-4 wks post-surgery or moderate, 4-8 wks post-surgery, OA was sufficient to decrease pain measures, with sustained improvement at later stages, after treatment was stopped.. Our data highlight the potential efficacy of antagonizing CCR2 at early stages to slow the progression of post-injury OA and, in addition, improve pain symptoms.

Topics: Animals; Benzoxazines; Bone and Bones; Cartilage, Articular; Chondrocytes; Disease Models, Animal; Disease Progression; Hypertrophy; Matrix Metalloproteinase 13; Menisci, Tibial; Mice; Osteoarthritis; Osteophyte; Receptors, CCR2; Sclerosis; Spiro Compounds; Tibial Meniscus Injuries

The repeated administration of microglial inhibitor (minocycline) and CCR2 antagonist (RS504393) attenuated the neuropathic pain symptoms in rats following chronic constriction injury of the sciatic nerve, which was associated with decreased spinal microglia activation and the protein level of CCL2 and CCR2. Furthermore, in microglia primary cell cultures minocycline downregulated both CCL2 and CCR2 protein levels after lipopolysaccharide-stimulation. Additionally, in astroglia primary cell cultures minocycline decreased the expression of CCL2, but not CCR2. Our results provide new evidence that modulation of CCL2/CCR2 pathway by microglial inhibitor as well as CCR2 antagonist is effective for neuropathic pain development in rats.

Topics: Analgesics; Animals; Animals, Newborn; Benzodiazepines; Benzoxazines; Cells, Cultured; Chemokine CCL2; Disease Models, Animal; Gene Expression Regulation; Hyperalgesia; Male; Minocycline; Neuroglia; Pain Threshold; Physical Stimulation; Rats; Rats, Wistar; Receptors, CCR2; Sciatica; Signal Transduction; Spinal Cord; Spiro Compounds

Interleukin (IL)-17 has been implicated in the pathogenesis of asthma and the progression of airway inflammation. Here, we used a model of allergic asthma and found that the frequencies of IL-17-secreting T helper (Th)17 and CD8 (Tc)17 cells were both significantly increased, as was the expression of the CC chemokine receptor (CCR2) on the surface of these cells. CC chemokine ligand 2 (CCL2) has been shown to mediate the activation and recruitment of inflammatory cells in asthma, which are also skewed after ovalbumin (OVA) challenge. However, the role of CCL2 on Th17 cells and Tc17 cells in asthma has not been illuminated.. Mice that were sensitized and challenged with OVA received anti-CCL2 antibody (Ab; 5 μg/day intratracheally) or CCR2 antagonist (RS504393, 2 mg/kg/day intraperitoneally) prior to the challenge. Some mice received an isotype control Ab or vehicle alone. We then assessed the effects of allergic asthma and anti-CCL2 Ab or CCR2 antagonist treatment on the levels of IL-17 and CCL2, the Th17 and Tc17 cell frequencies and lung tissue inflammation.. We demonstrated that CCL2 and IL-17 levels and the frequency of Th17 and Tc17 cells in lung tissues and bronchoalveolar lavage fluid increased in the asthma group compared with the normal control mice. Blocking the CCL2/CCR2 axis greatly reduced the Th17 but not the Tc17 cell frequency, and revealed a suppressive effect on airway inflammation.. These findings indicate a role for the CCL2/CCR2 axis in mediating Th17 but not Tc17 cell migration during acute allergic airway inflammation.

Topics: Animals; Antibodies, Neutralizing; Asthma; Benzoxazines; Bronchoalveolar Lavage Fluid; CD8-Positive T-Lymphocytes; Chemokine CCL2; Disease Models, Animal; Female; Gene Expression; Humans; Interleukin-17; Lung; Lymphocyte Count; Mice; Mice, Inbred BALB C; Ovalbumin; Receptors, CCR2; Spiro Compounds; Th17 Cells

Transforming growth factor α (TGFα) is increased in osteoarthritic (OA) cartilage in rats and humans and modifies chondrocyte phenotype. CCL2 is increased in OA cartilage and stimulates proteoglycan loss. This study was undertaken to test whether TGFα and CCL2 cooperate to promote cartilage degradation and whether inhibiting either reduces disease progression in a rat model of posttraumatic OA.. Microarray analysis was used to profile expression of messenger RNA (mRNA) for Tgfa, Ccl2, and related genes in a rat model of posttraumatic OA. Rat primary chondrocytes and articular cartilage explants were treated with TGFα in the presence or absence of MEK-1/2, p38, phosphatidylinositol 3-kinase, Rho-associated protein kinase, or CCR2 inhibitors and immunostained for markers of cartilage degradation. The rat model was used to administer pharmacologic inhibitors of TGFα (AG1478) and CCL2 (RS504393) signaling for up to 10 weeks and assess histopathology and serum biomarkers of cartilage synthesis (C-propeptide of type II collagen [CPII]) and breakdown (C2C).. Tgfa and Ccl2 mRNA were simultaneously up-regulated in articular cartilage in the rat model of posttraumatic OA. TGFα induced expression of CCL2, Mmp3, and Tnf in primary chondrocytes. Cleavage of type II collagen and aggrecan (by matrix metalloproteinases and ADAMTS-4/5, respectively) induced by TGFα was blocked by pharmacologic inhibition of CCL2 in cartilage explants. In vivo pharmacologic inhibition of TGFα or CCL2 signaling reduced Osteoarthritis Research Society International cartilage histopathology scores and increased serum CPII levels, but only TGFα inhibition reduced C2C levels intreated versus untreated rat OA cartilage.. TGFα signaling stimulates cartilage degradation via a CCL2-dependent mechanism, but pharmacologic inhibition of the TGFα-CCL2 axis reduces experimental posttraumatic OA progression in vivo.

Topics: Animals; Benzoxazines; Cartilage, Articular; Chemokine CCL2; Disease Models, Animal; Disease Progression; Male; Osteoarthritis; Quinazolines; Rats; Rats, Sprague-Dawley; Signal Transduction; Spiro Compounds; Transforming Growth Factor alpha; Tyrphostins; Up-Regulation; Wounds and Injuries

Lumbar disc herniation (LDH) is a major cause of sciatica, but the underlying mechanisms are not well understood. Chemokine CCL2 has been implicated to play a vital role in the neuroinflammation and central sensitization after spinal nerve ligation. Here we investigated the expression and the role of CCL2 and its receptor CCR2 in LDH-induced pain. Implantation of autologous nucleus pulposus induced persistent pain hypersensitivity, associated with increased mRNA expression of CCL2 and CCR2 in the dorsal root ganglion and spinal cord. Interestingly, CCL2 was increased in neurons and CCR2 was mainly increased in macrophages in the dorsal root ganglion, whereas CCL2 and CCR2 were increased in astrocytes and neurons, respectively, in the spinal cord. Intrathecal injection of CCR2 antagonist RS504393 at 3 days or 10 days significantly attenuated nucleus pulposus-induced mechanical allodynia. The results suggest that CCL2/CCR2 in the dorsal root ganglion and spinal cord is involved in the maintenance of LDH-induced pain. Targeting CCL2/CCR2 signaling may be a potential treatment for chronic radicular neuropathic pain.. These results suggest that CCL2/CCR2 signaling in the dorsal root ganglion and spinal cord is involved in LDH-induced pain via distinct mechanisms. These findings provide evidence of the antinociceptive effect of CCR2 antagonist on radicular neuropathic pain.

Topics: Analgesics; Animals; Astrocytes; Benzoxazines; Chemokine CCL2; Disease Models, Animal; Ganglia, Spinal; Hot Temperature; Hyperalgesia; Intervertebral Disc Displacement; Lumbar Vertebrae; Macrophages; Male; Neuralgia; Neurons; Rats, Sprague-Dawley; Receptors, CCR2; RNA, Messenger; Signal Transduction; Spinal Cord; Spiro Compounds; Touch

Methamphetamine addiction is characterized by drug craving caused by stimulation of the reward system. Because neuroinflammation underlies several neurological disorders, we investigated whether CC-chemokine ligand 2 (CCL2) participates in the methamphetamine dependence using mice. Upregulation of CCL2 but not CC-chemokine receptor 2 (CCR2), a dominant receptor for CCL2, mRNA in both the prefrontal cortex (PFC) and nucleus accumbens (NAC) was observed after methamphetamine (3 mg/kg, s.c.) administration. Using immunohistochemistry, high CCL2 protein levels localized to neurons in the PFC and NAC. In the conditioned place preference (CPP) test, methamphetamine (0.3 - 3 mg/kg, s.c.) induced a CPP, reflecting psychic dependence on methamphetamine, in a dose-dependent manner. The CPP to methamphetamine was attenuated by RS504393 (1 mg/kg, s.c.), a CCR2 antagonist. Moreover, methamphetamine increased phosphorylated tyrosine hydroxylase (pTH) levels in the ventral tegmental area (VTA). Increased levels of pTH in the VTA by methamphetamine was also suppressed by RS504393. Furthermore, intracerebroventricular injection of recombinant CCL2 increased pTH levels in the VTA. Taken together, we demonstrate that activation of dopamine neurons, which enhances reward-system activity, via the CCL2-CCR2 axis plays a crucial role in psychic dependence on methamphetamine. Novel treatments targeting this machinery may be effective for drug addiction.

Topics: Animals; Benzoxazines; Central Nervous System Stimulants; Chemokine CCL2; Disease Models, Animal; Dopaminergic Neurons; Dose-Response Relationship, Drug; Male; Methamphetamine; Mice; Mice, Inbred C57BL; Nucleus Accumbens; Prefrontal Cortex; Receptors, CCR2; Spiro Compounds; Substance Abuse Detection; Substance-Related Disorders; Tyrosine 3-Monooxygenase; Ventral Tegmental Area

Chemokines are proinflammatory mediators of the immune response, and there is growing evidence for chemokine/receptor signaling involvement in pronociception. Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain syndrome characterized by pain, pressure, or discomfort perceived to be bladder-related with at least one urinary symptom. We have explored the expression and functional roles of CCL2 (monocyte chemoattractant protein-1) and its high-affinity receptor, CCR2, in micturition reflex function and somatic sensitivity in rats with urinary bladder inflammation induced by cyclophosphamide (CYP) treatment of varying duration (4 h, 48 h, chronic). Real-time quantitative RT-PCR, ELISAs, and immunohistochemistry demonstrated significant (P ≤ 0.01) increases in CCL2 and CCR2 expression in the urothelium and in Fast Blue-labeled bladder afferent neurons in lumbosacral dorsal root ganglia with CYP-induced cystitis. Intravesical infusion of RS504393 (5 μM), a specific CCR2 antagonist, reduced voiding frequency and increased bladder capacity and void volume in rats with CYP-induced cystitis (4 h), as determined with open outlet, conscious cystometry. In addition, CCR2 blockade, at the level of the urinary bladder, reduced referred somatic sensitivity of the hindpaw and pelvic region in rats with CYP treatment, as determined with von Frey filament testing. We provide evidence of functional roles for CCL2/CCR2 signaling at the level of the urinary bladder in reducing voiding frequency and somatic sensitivity following CYP-induced cystitis (4 h). These studies suggest that chemokines/receptors may be novel targets with therapeutic potential in the context of urinary bladder inflammation.

Topics: Administration, Intravesical; Animals; Benzoxazines; Chemokine CCL2; Cyclophosphamide; Cystitis, Interstitial; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Ganglia, Spinal; Immunohistochemistry; Neurons, Afferent; Pain Measurement; Pain Threshold; Rats; Rats, Wistar; Real-Time Polymerase Chain Reaction; Receptors, CCR2; Reflex; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Spiro Compounds; Time Factors; Up-Regulation; Urinary Bladder; Urination; Urodynamics

The participation of the chemokine CCL2 (monocyte chemoattractant protein-1) in inflammatory and neuropathic pain is well established. Furthermore, the release of CCL2 from a NCTC 2472 cells-evoked tumor and its involvement in the upregulation of calcium channel α2δ1 subunit of nociceptors was demonstrated. In the present experiments, we have tried to determine whether the increase in CCL2 levels is a common property of painful tumors and, in consequence, the administration of a chemokine receptor type 2 (CCR2) antagonist can inhibit tumoral hypernociception. CCL2 levels were measured by ELISA in the tumoral region of mice intratibially inoculated with NCTC 2472 or B16-F10 cells, and the antihyperalgesic and antiallodynic effects evoked by the administration of the selective CCR2 antagonist RS 504393 were assessed. Cultured NCTC 2472 cells release CCL2 and their intratibial inoculation evokes the development of a tumor in which CCL2 levels are increased. Moreover, the systemic or peritumoral administration of RS 504393 inhibited thermal and mechanical hyperalgesia, but not mechanical allodynia evoked after the inoculation of these cells. Thermal hyperalgesia was also inhibited by the peritumoral administration of a neutralizing CCL2 antibody. In contrast, no change in CCL2 levels was observed in mice inoculated with B16-F10 cells, and RS 504393 did not inhibit the hypernociceptive reactions evoked by their intratibial inoculation. The peripheral release of CCL2 is involved in the development of thermal and mechanical hyperalgesia, but not mechanical allodynia evoked by the inoculation of NCTC 2472 cells, whereas this chemokine seems unrelated to the hypernociception induced by B16-F10 cells.

Topics: Animals; Benzoxazines; Bone Neoplasms; Cell Line, Tumor; Chemokine CCL2; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Fibrosarcoma; Hyperalgesia; Male; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Receptors, CCR2; Spiro Compounds; Tibia